what is a two hit hypothesis

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Knudson's "Two-Hit" Theory of Cancer Causation

The "two-hit" hypothesis provided a unifying model for understanding cancer that occurs in individuals who carry a "susceptibility gene" and cancers that develop because of randomly induced mutations in otherwise normal genes. Tumor-suppressor genes, in particular, are important targets for cancer prevention research, since they normally function to apply the brakes to cellular growth.

Like many significant conceptual leaps in science, Knudson's "two-hit" hypothesis was met with skepticism when he first published it in 1971, yet Knudson's powerful insights into the development of cancer hold implications for both cancer treatment and prevention.

Defects in tumor-suppressor genes permit abnormal, cancerous growth, so devising ways to remedy such flaws or replace the gene's missing product through medication are of interest to researchers. 

Alfred G. Knudson Jr., MD, PhD

A geneticist and physician, Dr. Knudson (August 9, 1922 – July 10, 2016) was internationally recognized for his "two-hit" theory of cancer causation, which explained the relationship between the hereditary and non-hereditary forms of a cancer and predicted the existence of tumor-suppressor genes that can suppress cancer cell growth. This now-confirmed theory has advanced understanding of errors in the genetic program that turn normal cells into cancer cells.

Distinguished Awards

Among Knudson's many professional distinctions, he received the 2004 Kyoto Prize , considered among the world's leading awards for lifetime achievement. He also earned the 1998 Albert Lasker Award for Clinical Medical Research , one of seven Lasker Awards presented that year. Considered "America's Nobels," Lasker Awards rank among the highest recognition for careers of distinguished work because of the extremely rigorous process of nomination and selection conducted by a jury of the world's top scientists.

In 1999, Knudson received the Distinguished Career Award of the American Society of Hematology/Oncology and the international John Scott Award from the City of Philadelphia. In 2000, the American Academy of Dermatology honored him with its Lila Gruber Memorial Cancer Research Award for researchers whose lifetime contributions have been outstanding in importance and distinction.

When Dr. Knudson became an AACR honorary member in 2011 he reviewed his distinguished career, and his most significant accomplishments that laid the groundwork for the tumor suppressor concept, in an interview with AACR News.

In September 2002, Knudson received Fox Chase Cancer Center's 14th annual Wick R. Williams Memorial Award. The American Society of Clinical Oncology also honored him with its 2002 Special Award in the form of a Pediatric Oncology Lectureship recognizing individuals who are accomplished in pediatric oncology.

In addition, Knudson has received the 1988 Charles S. Mott Prize of the General Motors Cancer Research Foundation; the American Cancer Society's 1989 Medal of Honor; the 1990 Founders' Award of the Chemical Industry Institute for Toxicology; the American Radium Society's 1990 Janeway Medal; Memorial Sloan-Kettering Cancer Center's 1990 Katharine Berkan Judd Award; the 1991 William Allan Memorial Award of the American Society of Human Genetics; M. D. Anderson Cancer Center's 1995 Bertner Award; Switzerland's 1995 Charles Rodolphe Brupbacher Foundation Award; the 1996 Robert J. and Claire Pasarow Foundation Award; the 1996 Durham City of Medicine Award; Canada's 1997 Gairdner Foundation International Award; and the American Society of Clinical Oncology's 1997 Karnofsky Memorial Lecture Award.

Knudson's Background and Education

Born in Los Angeles in 1922, Knudson received his BS from California Institute of Technology in 1944, his MD from Columbia University in 1947 and his PhD from California Institute of Technology in 1956. He held a Guggenheim fellowship from 1953 to 1954.

Knudson came to Fox Chase from the University of Texas Graduate School of Biomedical Sciences, where he was dean, and the M.D. Anderson Hospital and Tumor Institute in Houston, Texas, where he specialized in pediatrics and biology. Previously, he was associate dean for basic sciences at the State University of New York at Stony Brook from 1966 to 1969. He began his affiliation with Fox Chase in 1970 as a member of its scientific advisory committee before joining the Center staff in 1976.

Knudson and his wife, Anna T. Meadows, MD, a pediatric oncologist at Children's Hospital of Philadelphia, collaborated on the study of the genetics of childhood cancer.

He died July 10, 2016.

Career Highlights

Dr. Knudson became an honorary member of the American Association for Cancer Research (AACR) in 2011, and an inaugural Fellow of the AACR Academy  in March 2013. 

In honor of his contributions to biomedical science, Knudson has been elected to the National Academy of Sciences and was named a Fox Chase Distinguished Scientist and senior advisor to the president in 1992. He was instrumental as a leader of Fox Chase's molecular oncology program from 1989 to 1999. Previously, Knudson served as director of Fox Chase's Institute for Cancer Research from 1976 until 1982, Center president from 1980 to 1982 and scientific director from 1982 to 1983.

In 1995, Knudson was appointed as special advisor to Dr. Richard Klausner, then director of the National Cancer Institute. While continuing his work at Fox Chase, Knudson also worked closely with Dr. Joseph Fraumeni in NCI's Division of Cancer Epidemiology and Genetics. Knudson served as acting director of its human genetics program until September 1999, when he returned to Fox Chase full-time.

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The Two-Hit Hypothesis Meets Epigenetics

Jean-pierre issa.

Coriell Institute for Medical Research, Camden, New Jersey.

The landmark paper by Kane and colleagues was the first report of DNA methylation in the promoter of the human MLH1 gene in sporadic colon cancers with mismatch repair (MMR) deficiency. In both cell lines and primary tumors, promoter methylation was associated with loss of MLH1 protein expression and with a lack of mutations in the MLH1 coding region. Together with subsequent papers that showed that this methylation was directly responsible for loss of MLH1 expression and MMR deficiency, the observation expanded the two-hit hypothesis of tumor suppressor gene loss in cancer to include both genetic and epigenetic mechanisms of gene inactivation. More broadly, the paper contributed to normalization of the hypothesis of an epigenetic basis for cancer development.

Cancer: Genetic or Epigenetic Disease?

The idea of cancer as a disease of epigenetic regulation was proposed in the 1960’s based on unexpected reversal of the cancer phenotype when malignant cells undergo epigenetic reprogramming though embryogenesis ( 1 ). Epigenetic mechanisms were still mysterious at the time, and in the absence of a molecular explanation, the concept was rapidly forgotten. By the 1970’s, studies of familial cancer incidence had firmly established a genetic basis for inherited cancer, and it was hypothesized that sporadic cancers would follow the same path. Knudson formulated the two-hit hypothesis to explain familial versus sporadic cancer epidemiology, proposing that the same genes are involved in both cases ( 2 ). This was confirmed molecularly with studies on familial cancer genes such as TP53 ( 3 ). The dogma evolved to a model whereby the two hits are mutations or deletions in the same tumor suppressor gene (TSG; Fig. 1 ); familial cases would be explained by an inherited hit followed by a sporadic hit, while nonfamilial cases are caused by two rare sporadic hits, explaining the delayed age of onset when compared with familial cancers ( 2 ).

The two-hit hypothesis meets epigenetics. By the 1990’s, Knudson’s two-hit hypothesis had evolved to postulate that familial cancer predisposition is due to a germline mutation in a TSG (top left), while actual cancer development follows a sporadic mutation (or deletion) in the second allele (top right). It also postulated that sporadic cancers of the same tissue type were due to two acquired hits (mutation or deletion) in the same TSG (bottom). The paper by Kane and colleagues (and other papers in that period) revised that model for sporadic cancers to include epigenetic inactivation by promoter DNA hypermethylation as either the first hit, the second hit, or both (bottom right). The figure is a simplification; subsequent data showed that in very rare cases familial cancers can be caused by germline hypermethylation, that the second hit in familial cancers can also be DNA hypermethylation, and, in the case of MLH1 in colon cancer, the “hits” in sporadic cases are almost always hypermethylation caused by the CpG island methylator phenotype.

Inherited mutations in MLH1 cause the Lynch syndrome, a familiar colon cancer predisposition disease ( 3 ). Biallelic inactivation of MLH1 causes mismatch repair (MMR) deficiency, accumulation of mutations, and eventual cancer formation. The phenotype of MMR deficiency was known before the genotype was elucidated (it was termed microsatellite instability or MSI at the time), and it was clear early on that MMR defects were present in both familial and sporadic colon cancers ( 4 ). In accordance with Knudson’s hypotheses, it was expected that the genes that cause familial versus sporadic MMR-deficient cancers would be the same. Kane and colleagues tried to confirm this ( 4 ). Unexpectedly, while they found that sporadic MSI+ cases had loss of MLH1 protein, they could find no coding sequence mutations in this gene, challenging the prevailing notion at the time.

In the 1980’s, the unorthodox idea of an epigenetic basis for cancer resurfaced, powered by an actual proposed mechanism—permanent silencing of TSG expression by promoter DNA methylation ( 5 ). As besets many “out-of-the-box” hypotheses, this one was met with considerable early skepticism. Nevertheless, after Kane and colleagues observed unexplained loss of MLH1 expression in colon cancers, they went on to show that those sporadic cell lines and primary tumors that lacked MLH1 expression also had dense DNA methylation in a CpG rich area in the gene promoter ( 4 ). This observation was therefore consistent with Knudson’s tenets, but with a twist—the gene in sporadic and familial cancers was the same ( MLH1 ) but the mechanism of inactivation was different (DNA methylation vs. mutations; Fig. 1 ). The fact that MLH1 was a bona fide cancer predisposition gene (a “gatekeeper” in Vogelstein’s model; ref. 3 ) inactivated by DNA methylation was a turning point in the wide acceptance of the epigenetic basis of cancer hypothesis. It was further buttressed by the demonstration that the MMR phenotype can be reversed by removal of DNA methylation ( 6 ) and also by the identification of other TSGs with similar genetic/epigenetic mechanisms of inactivation ( 5 ). As can be seen in cancer biology textbooks 25 years later, the paper by Kane and colleagues contributed to rewriting the chapters on the mechanistic basis of cancer development—cancer is now seen as a genetic and an epigenetic disease.

The Two-Hit Hypothesis Revised

An important consequence of the paper by Kane and colleagues (and other papers at the time) was the modification of the two-hit hypothesis to include both genetic and epigenetic mechanisms of gene inactivation ( Fig. 1 ). Because epigenetic changes are reset during embryogenesis, it is not surprising that, with rare (and fascinating) exceptions, all familial cancers are caused by genetic changes. For reasons not yet fully understood, some TSGs are resistant to epigenetic inactivation. In those cases, the second hit in familial cancers as well as both hits in sporadic cancers are also genetic. For the other TSGs (such as MLH1 , VHL , CDKN2A , BRCA1 , and others), the second hit in familial cases can be either genetic or epigenetic, and both hits in sporadic cases can similarly be either genetic or epigenetic. Lynch syndrome provides an interesting study in this genetic/epigenetic duality. The MSH2 gene is a frequent cause of familial disease but is almost never epigenetically inactivated, and it is very rarely responsible for sporadic colon cancers. By contrast, the MLH1 gene is also a frequent cause of familial disease, but it is susceptible to epigenetic inactivation and is almost always responsible for MMR-deficient sporadic colon cancers.

Cycles of Instability

In addition to revising the two-hit hypothesis, the paper by Kane and colleagues suggested that epigenetic changes can trigger genetic changes by inducing DNA repair defects. In an interesting additional twist, MLH1 methylation in sporadic cancers was subsequently shown to be caused by a broader epigenetic deregulation, the CpG island methylator phenotype (CIMP; ref. 7 ). CIMP+ cancers inactivate multiple TSGs simultaneously, and this phenotype of epigenetic instability has been described in almost all cancer cell types ( 7 ). Intriguingly, MLH1 is a common CIMP target in colon and endometrial cancers, but not in other CIMP+ cases. In acute myelogenous leukemia (AML) and in glioblastoma multiforme (GBM), CIMP is caused by genetic defects in the TCA cycle, namely mutations in IDH1 or IDH2 ( 8 ). These mutations lead to accumulation of metabolites that inactivate the TET enzymes, which are responsible for protection against DNA methylation in CpG islands ( 8 ). Thus, mutations in metabolic regulation genes can lead to epigenetic instability. In turn, epigenetic instability can lead to genetic instability by inducing DNA repair defects ( 7 ). As one can imagine, these cycles of instability can be repeated many times in the lifetime of a cancer, contributing to progression, metastasis, and therapeutic resistance.

Unlike AML and GBM, mutations in TCA cycle genes have not been commonly seen in CIMP+ colon cancers ( 9 ) or gastric cancers. Even in AML, CIMP can only be explained by mutations in about half the cases. The etiology of CIMP in mutation-negative cases remains speculative. There is a strong association between CIMP and Epstein-Barr virus in gastric cancers ( 7 ), and an intriguing link between CIMP and fusobacterium in colon cancers ( 9 ), suggesting an environmental etiology to CIMP in some cases. It is possible for example that infections lead to metabolic deregulation, disrupting TET function and leading to DNA hypermethylation.

Clinical Implications

The diagnosis of a familial cancer syndrome can save lives through early screening and intervention. Lynch syndrome has an incomplete penetrance and can first manifest at an advanced age. While colon cancers are routinely screened for MMR defects, a positive result does not always trigger an investigation of germline defects because the frequency of sporadic MMR-deficient cancers is higher than that of familial MMR-deficient cancers after the age of 60. The paper by Kane and colleagues (and subsequent confirmation) suggests that MLH1 methylation can serve as a quick additional screen in this respect. MMR-deficient colon cancers that lack MLH1 expression but have no MLH1 promoter DNA methylation should trigger a very high index of suspicion for Lynch syndrome.

Finally, it is worth noting that epigenetic changes mediated by DNA methylation are potentially reversible through the use of drugs that target DNA methyltransferases ( 10 ). The paper by Kane and colleagues ( 4 ) and other papers showing the importance of DNA methylation in TSG regulation in cancer ( 5 ) triggered a revival in interest in these drugs, eventually leading to their FDA approval and broad usage in patients with myeloid malignancies ( 10 ). While their activity in solid tumors remains limited, there continues to be interest in developing DNA methylation–based treatment or prevention strategies that could be especially useful in cases with CIMP and MLH1 promoter DNA methylation.

Conclusions

The paper by Kane and colleagues had a straightforward message: MLH1 expression was missing in MMR-deficient sporadic colon cancers and this was associated with promoter DNA methylation rather than coding mutations in the gene ( 4 ). This deceptively simple message had a profound influence in that it lent legitimacy to the hypothesis of DNA methylation–mediated TSG inactivation in cancer. It helped rewrite textbooks—“Cancer is a genetic and an epigenetic disease.”

Acknowledgments

The author is funded by the NCI R01 CA158112, P50 CA100632, and P50 CA254897. The author would also like to acknowledge the many scientists who contributed to generating the concepts described in the commentary but whose primary papers could not be cited due to space constraints.

Authors’ Disclosures

J.J. Issa reports personal fees from Ascentage Pharma and personal fees from Daiichi Sankyo outside the submitted work.

• November 1999

'Two-Hit' Hypothesis

Much of what scientists know about the origins of cancer and the role of tumor suppressors can be traced back 28 years to the elegant theory of cancer researcher alfred g. knudson. widely thought to be one of the most significant theories in modern biology, knudson's "two-hit" hypothesis was recognized nov. 19 at the john scott awards in philadelphia, along with the revolutionary research of benoit mandelbrot, the discoverer of the powerful mathematical laws governing fractal geometry and self-s.

Much of what scientists know about the origins of cancer and the role of tumor suppressors can be traced back 28 years to the elegant theory of cancer researcher Alfred G. Knudson . Widely thought to be one of the most significant theories in modern biology, Knudson's "two-hit" hypothesis was recognized Nov. 19 at the John Scott Awards in Philadelphia, along with the revolutionary research of Benoit Mandelbrot , the discoverer of the powerful mathematical laws governing fractal geometry and self-similarity. 1

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• Two hits revisited again
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• I P M Tomlinson a ,
• R Roylance a ,
• R S Houlston b
• a Molecular and Population Genetics Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, UK, b Section of Cancer Genetics, Institute of Cancer Research, Cotswold Road, Sutton, Surrey SM2 5NG, UK
• Dr Tomlinson, i.tomlinson{at}icrf.icnet.uk

INTRODUCTION AND METHODS Since the concept of the “two hit hypothesis” was introduced over 20 years ago, a wealth of genetic data has accumulated on the mutations found at tumour suppressor loci. Perhaps surprisingly, these data conceal large gaps in our knowledge which genetic and functional studies are beginning to uncover. The “two hit hypothesis” must be updated to take account of this new information.

RESULTS AND DISCUSSION Here, we discuss both the results of recent studies and some of the questions that they highlight. In particular, how valid are conclusions from inherited Mendelian syndromes when applied to sporadic cancers? Why is allelic loss so common and how does it occur? Are the “two hits” random or interdependent? Is abolition of protein function always optimal for tumorigenesis? Can “third hits” occur and, if so, why? How can mismatch repair deficiency and the methylator phenotype be incorporated into the “two hit” hypothesis? We suggest that the “two hit hypothesis” is not fixed but is evolving as our knowledge expands.

• two hit model
• tumour suppressor
• carcinogenesis

https://doi.org/10.1136/jmg.38.2.81

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Hereditary cancer: two hits revisited

Affiliation.

• 1 Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA USA.
• PMID: 8601560
• DOI: 10.1007/BF01366952

According to a "two-hit" model, dominantly inherited predisposition to cancer entails a germline mutation, while tumorigenesis requires a second, somatic, mutation. Non-hereditary cancer of the same type requires the same two hits, but both are somatic. The original tumor used in this model, retinoblastoma, involves mutation or loss of both are somatic. The original tumor used in this model, retinoblastoma, involves mutation or loss of both copies of the RB1 tumor-suppressor gene in both hereditary and non-hereditary forms. In fact, most dominantly inherited cancers show this relationship. New dominantly inherited cancers show this relationship. New questions have arisen, however. When a tumor-suppressor gene is ubiquitously expressed, why is there any specificity of tumor predilection? In some instances, it is clear that two hits produce only a benign precursor lesion and that other genetic events are necessary. As the number of necessary events increase, the impact of the germline mutation diminishes. The number of events is least for embryonal tumors, and relatively small for certain sarcomas. Stem-cell proliferation evidently plays a key role early in carcinogenesis. In some tissues it is physiological, as in embryonic development and in certain tissues in adolescence. In adult renewal tissues, the sites of the common carcinomas, mutation may be necessary to impair the control of switching between renewal and replicative cell divisions; the APC gene may be the target of such a mutation.

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Cancer: Genetic or Epigenetic Disease?

The two-hit hypothesis revised, cycles of instability, clinical implications, conclusions, authors' disclosures, acknowledgments, the two-hit hypothesis meets epigenetics.

Cancer Res 2022;82:1167–9

• Funder(s):  NCI
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Jean-Pierre Issa; The Two-Hit Hypothesis Meets Epigenetics. Cancer Res 1 April 2022; 82 (7): 1167–1169. https://doi.org/10.1158/0008-5472.CAN-22-0405

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The landmark paper by Kane and colleagues was the first report of DNA methylation in the promoter of the human MLH1 gene in sporadic colon cancers with mismatch repair (MMR) deficiency. In both cell lines and primary tumors, promoter methylation was associated with loss of MLH1 protein expression and with a lack of mutations in the MLH1 coding region. Together with subsequent papers that showed that this methylation was directly responsible for loss of MLH1 expression and MMR deficiency, the observation expanded the two-hit hypothesis of tumor suppressor gene loss in cancer to include both genetic and epigenetic mechanisms of gene inactivation. More broadly, the paper contributed to normalization of the hypothesis of an epigenetic basis for cancer development.

See related article by Kane and colleagues, Cancer Res 1997;57:808–11

The idea of cancer as a disease of epigenetic regulation was proposed in the 1960's based on unexpected reversal of the cancer phenotype when malignant cells undergo epigenetic reprogramming though embryogenesis ( 1 ). Epigenetic mechanisms were still mysterious at the time, and in the absence of a molecular explanation, the concept was rapidly forgotten. By the 1970's, studies of familial cancer incidence had firmly established a genetic basis for inherited cancer, and it was hypothesized that sporadic cancers would follow the same path. Knudson formulated the two-hit hypothesis to explain familial versus sporadic cancer epidemiology, proposing that the same genes are involved in both cases ( 2 ). This was confirmed molecularly with studies on familial cancer genes such as TP53 ( 3 ). The dogma evolved to a model whereby the two hits are mutations or deletions in the same tumor suppressor gene (TSG; Fig. 1 ); familial cases would be explained by an inherited hit followed by a sporadic hit, while nonfamilial cases are caused by two rare sporadic hits, explaining the delayed age of onset when compared with familial cancers ( 2 ).

The two-hit hypothesis meets epigenetics. By the 1990's, Knudson's two-hit hypothesis had evolved to postulate that familial cancer predisposition is due to a germline mutation in a TSG (top left), while actual cancer development follows a sporadic mutation (or deletion) in the second allele (top right). It also postulated that sporadic cancers of the same tissue type were due to two acquired hits (mutation or deletion) in the same TSG (bottom). The paper by Kane and colleagues (and other papers in that period) revised that model for sporadic cancers to include epigenetic inactivation by promoter DNA hypermethylation as either the first hit, the second hit, or both (bottom right). The figure is a simplification; subsequent data showed that in very rare cases familial cancers can be caused by germline hypermethylation, that the second hit in familial cancers can also be DNA hypermethylation, and, in the case of MLH1 in colon cancer, the “hits” in sporadic cases are almost always hypermethylation caused by the CpG island methylator phenotype.

Inherited mutations in MLH1 cause the Lynch syndrome, a familiar colon cancer predisposition disease ( 3 ). Biallelic inactivation of MLH1 causes mismatch repair (MMR) deficiency, accumulation of mutations, and eventual cancer formation. The phenotype of MMR deficiency was known before the genotype was elucidated (it was termed microsatellite instability or MSI at the time), and it was clear early on that MMR defects were present in both familial and sporadic colon cancers ( 4 ). In accordance with Knudson's hypotheses, it was expected that the genes that cause familial versus sporadic MMR-deficient cancers would be the same. Kane and colleagues tried to confirm this ( 4 ). Unexpectedly, while they found that sporadic MSI+ cases had loss of MLH1 protein, they could find no coding sequence mutations in this gene, challenging the prevailing notion at the time.

In the 1980's, the unorthodox idea of an epigenetic basis for cancer resurfaced, powered by an actual proposed mechanism—permanent silencing of TSG expression by promoter DNA methylation ( 5 ). As besets many “out-of-the-box” hypotheses, this one was met with considerable early skepticism. Nevertheless, after Kane and colleagues observed unexplained loss of MLH1 expression in colon cancers, they went on to show that those sporadic cell lines and primary tumors that lacked MLH1 expression also had dense DNA methylation in a CpG rich area in the gene promoter ( 4 ). This observation was therefore consistent with Knudson's tenets, but with a twist—the gene in sporadic and familial cancers was the same ( MLH1 ) but the mechanism of inactivation was different (DNA methylation vs. mutations; Fig. 1 ). The fact that MLH1 was a bona fide cancer predisposition gene (a “gatekeeper” in Vogelstein's model; ref. 3 ) inactivated by DNA methylation was a turning point in the wide acceptance of the epigenetic basis of cancer hypothesis. It was further buttressed by the demonstration that the MMR phenotype can be reversed by removal of DNA methylation ( 6 ) and also by the identification of other TSGs with similar genetic/epigenetic mechanisms of inactivation ( 5 ). As can be seen in cancer biology textbooks 25 years later, the paper by Kane and colleagues contributed to rewriting the chapters on the mechanistic basis of cancer development—cancer is now seen as a genetic and an epigenetic disease.

An important consequence of the paper by Kane and colleagues (and other papers at the time) was the modification of the two-hit hypothesis to include both genetic and epigenetic mechanisms of gene inactivation ( Fig. 1 ). Because epigenetic changes are reset during embryogenesis, it is not surprising that, with rare (and fascinating) exceptions, all familial cancers are caused by genetic changes. For reasons not yet fully understood, some TSGs are resistant to epigenetic inactivation. In those cases, the second hit in familial cancers as well as both hits in sporadic cancers are also genetic. For the other TSGs (such as MLH1 , VHL , CDKN2A , BRCA1 , and others), the second hit in familial cases can be either genetic or epigenetic, and both hits in sporadic cases can similarly be either genetic or epigenetic. Lynch syndrome provides an interesting study in this genetic/epigenetic duality. The MSH2 gene is a frequent cause of familial disease but is almost never epigenetically inactivated, and it is very rarely responsible for sporadic colon cancers. By contrast, the MLH1 gene is also a frequent cause of familial disease, but it is susceptible to epigenetic inactivation and is almost always responsible for MMR-deficient sporadic colon cancers.

In addition to revising the two-hit hypothesis, the paper by Kane and colleagues suggested that epigenetic changes can trigger genetic changes by inducing DNA repair defects. In an interesting additional twist, MLH1 methylation in sporadic cancers was subsequently shown to be caused by a broader epigenetic deregulation, the CpG island methylator phenotype (CIMP; ref. 7 ). CIMP+ cancers inactivate multiple TSGs simultaneously, and this phenotype of epigenetic instability has been described in almost all cancer cell types ( 7 ). Intriguingly, MLH1 is a common CIMP target in colon and endometrial cancers, but not in other CIMP+ cases. In acute myelogenous leukemia (AML) and in glioblastoma multiforme (GBM), CIMP is caused by genetic defects in the TCA cycle, namely mutations in IDH1 or IDH2 ( 8 ). These mutations lead to accumulation of metabolites that inactivate the TET enzymes, which are responsible for protection against DNA methylation in CpG islands ( 8 ). Thus, mutations in metabolic regulation genes can lead to epigenetic instability. In turn, epigenetic instability can lead to genetic instability by inducing DNA repair defects ( 7 ). As one can imagine, these cycles of instability can be repeated many times in the lifetime of a cancer, contributing to progression, metastasis, and therapeutic resistance.

Unlike AML and GBM, mutations in TCA cycle genes have not been commonly seen in CIMP+ colon cancers ( 9 ) or gastric cancers. Even in AML, CIMP can only be explained by mutations in about half the cases. The etiology of CIMP in mutation-negative cases remains speculative. There is a strong association between CIMP and Epstein-Barr virus in gastric cancers ( 7 ), and an intriguing link between CIMP and fusobacterium in colon cancers ( 9 ), suggesting an environmental etiology to CIMP in some cases. It is possible for example that infections lead to metabolic deregulation, disrupting TET function and leading to DNA hypermethylation.

The diagnosis of a familial cancer syndrome can save lives through early screening and intervention. Lynch syndrome has an incomplete penetrance and can first manifest at an advanced age. While colon cancers are routinely screened for MMR defects, a positive result does not always trigger an investigation of germline defects because the frequency of sporadic MMR-deficient cancers is higher than that of familial MMR-deficient cancers after the age of 60. The paper by Kane and colleagues (and subsequent confirmation) suggests that MLH1 methylation can serve as a quick additional screen in this respect. MMR-deficient colon cancers that lack MLH1 expression but have no MLH1 promoter DNA methylation should trigger a very high index of suspicion for Lynch syndrome.

Finally, it is worth noting that epigenetic changes mediated by DNA methylation are potentially reversible through the use of drugs that target DNA methyltransferases ( 10 ). The paper by Kane and colleagues ( 4 ) and other papers showing the importance of DNA methylation in TSG regulation in cancer ( 5 ) triggered a revival in interest in these drugs, eventually leading to their FDA approval and broad usage in patients with myeloid malignancies ( 10 ). While their activity in solid tumors remains limited, there continues to be interest in developing DNA methylation–based treatment or prevention strategies that could be especially useful in cases with CIMP and MLH1 promoter DNA methylation.

The paper by Kane and colleagues had a straightforward message: MLH1 expression was missing in MMR-deficient sporadic colon cancers and this was associated with promoter DNA methylation rather than coding mutations in the gene ( 4 ). This deceptively simple message had a profound influence in that it lent legitimacy to the hypothesis of DNA methylation–mediated TSG inactivation in cancer. It helped rewrite textbooks—“Cancer is a genetic and an epigenetic disease.”

J.J. Issa reports personal fees from Ascentage Pharma and personal fees from Daiichi Sankyo outside the submitted work.

The author is funded by the NCI R01 CA158112, P50 CA100632, and P50 CA254897. The author would also like to acknowledge the many scientists who contributed to generating the concepts described in the commentary but whose primary papers could not be cited due to space constraints.

commentary-article

• Methylation of the hMLH1 Promoter Correlates with Lack of Expression of hMLH1 in Sporadic Colon Tumors and Mismatch Repair-defective Human Tumor Cell Lines 1

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• Oncogenomics
• Published: 06 October 2008

APC and the three-hit hypothesis

• S Segditsas 1   na1 ,
• A J Rowan 1   na1 ,
• K Howarth 1   na1 ,
• A Jones 1 ,
• S Leedham 2 ,
• N A Wright 2 ,
• P Gorman 1 ,
• W Chambers 1 , 3 ,
• E Domingo 1 ,
• R R Roylance 1 ,
• E J Sawyer 1 ,
• O M Sieber 1   nAff5 &
• I P M Tomlinson 1  

Oncogene volume  28 ,  pages 146–155 ( 2009 ) Cite this article

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The seminal ‘two-hit hypothesis’ implicitly assumes that bi-allelic tumour suppressor gene (TSG) mutations cause loss of protein function. All subsequent events in that tumour therefore take place on an essentially null background for that TSG protein. We have shown that the two-hit model requires modification for the APC TSG, because mutant APC proteins probably retain some function and the two hits are co-selected to produce an optimal level of Wnt activation. We wondered whether the optimal Wnt level might change during tumour progression, leading to selection for more than two hits at the APC locus. Comprehensive screening of a panel of colorectal cancer (CRC) cell lines and primary CRCs showed that some had indeed acquired third hits at APC . These third hits were mostly copy number gains or deletions, but could be protein-truncating mutations. Third hits were significantly less common when the second hit at APC had arisen by copy-neutral loss of heterozygosity. Both polyploid and near-diploid CRCs had third hits, and the third hits did not simply arise as a result of acquiring a polyploid karyotype. The third hits affected mRNA and protein levels, with potential functional consequences for Wnt signalling and tumour growth. Although some third hits were probably secondary to genomic instability, others did appear specifically to target APC . Whilst it is generally believed that tumours develop and progress through stepwise accumulation of mutations in different functional pathways, it also seems that repeated targeting of the same pathway and/or gene is selected in some cancers.

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Acknowledgements

We are grateful to colleagues at St Mark's Hospital for tissue collection, to several kind providers of cell lines and to the Mutation Detection Facility, Cancer Research UK London Research Institute.

Author information

Present address: 5Current address: Ludwig Colon Cancer Initiative Laboratory, Ludwig Institute for Cancer Research, PO Box 2008, Royal Melbourne Hospital, VIC 3050, Australia,

S Segditsas, A J Rowan and K Howarth: These authors contributed equally to this work.

Authors and Affiliations

Molecular and Population Genetics Laboratory, London Research Institute, Cancer Research UK, London, UK

S Segditsas, A J Rowan, K Howarth, A Jones, P Gorman, W Chambers, E Domingo, R R Roylance, E J Sawyer, O M Sieber & I P M Tomlinson

Histopathology Laboratory, London Research Institute, Cancer Research UK, London, UK

S Leedham & N A Wright

Department of Colorectal Surgery and Genetics Knowledge Park, Oxford Radcliffe Hospitals, Oxford, UK

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Correspondence to I P M Tomlinson .

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Segditsas, S., Rowan, A., Howarth, K. et al. APC and the three-hit hypothesis. Oncogene 28 , 146–155 (2009). https://doi.org/10.1038/onc.2008.361

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Received : 20 February 2008

Revised : 07 July 2008

Accepted : 28 August 2008

Published : 06 October 2008

Issue Date : 08 January 2009

DOI : https://doi.org/10.1038/onc.2008.361

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